The present invention relates to the use of lactic acid bacteria to reduce lactose in dairy products and their use in food and other commercial applications.
Lactose, or milk sugar, is a disaccharide carbohydrate that is hydrolyzed to glucose and galactose by the enzyme lactase, also known as beta-galactosidase (xcex2-galactosidase). Although lactase is normally present in humans in intestinal epithelial cells and thereby contributes to the digestion of lactose present in dairy foods, a significant number of the adult human population is unable to digest lactose due to a genetic deficiency of this enzyme. Persons deficient in lactase may react to the consumption of dairy products with symptoms including nausea, cramps, gas, and diarrhea, a clinical condition known as lactose intolerance. Temporary lactase deficiencies in individuals with normal lactase levels may also result from damage to the intestinal lining produced by viral or bacterial infections, cancer chemotherapy, allergic or autoimmune conditions, and from decreases in lactase associated with aging. Persons who are lactase deficient and wish to consume normal dairy products with high lactose levels such as milk (12-13 grams of lactose per glass) can either purchase lactase treated dairy foods (70-100% hydrolyzed lactose) or consume a dietary supplement of lactase enzyme before each dairy meal. The cost and properties of current lactase preparations have limited the availability of lactose-reduced or free dairy products and other foods. Improvements in enzyme production costs and hydrolysis reaction properties could lead to the availability of a larger number of lactose reduced and free dairy foods and ingredients as well as more effective prophylactic products.
The invention is based, in part, on the discovery of a method of achieving enhanced lactose hydrolysis using lactic acid bacteria. More specifically, the invention relates to a method of hydrolyzing lactose by using lactic acid bacteria that produce high levels of xcex2-galactosidase, permeablizing the bacteria such that lactose can enter the cell and be hydrolyzed by the highly concentrated xcex2-galactosidase contained therein. The above method results in the generation of permeablized lactic acid bacteria which contain high concentrations of xcex2-galactosidase (referred to herein as lactase microcarriers) and which have a surprisingly high ability to rapidly and efficiently hydrolyze lactose under a variety of conditions. Moreover, the lactase microcarriers also have protease and bile resistant properties making them an ideal supplemental enzyme and oral prophylactic against the clinical condition of lactose intolerance.
Accordingly, the invention features a method for preparing a lactase microcarrier for hydrolyzing lactose in a liquid, e.g., milk, a whey product, or derivatives thereof. The method includes transforming a food-grade lactic acid bacterium with a DNA construct, wherein the DNA construct includes a promoter sequence of a gene operatively linked to a DNA sequence encoding a xcex2-galactosidase, culturing the transformed bacterium under conditions that enable expression of the xcex2-galactosidase such that the xcex2-galactosidase activity exhibited in the bacterium is at least about 4000 MU (e.g., at least 4500, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000 or 15,000 MU), and permeabilizing the bacterium. The method can further include contacting the permeabilized bacterium with a liquid containing lactose for a time sufficient to hydrolyze the lactose. The lactic acid bacterium can be a Streptococcus, Aerococcus, Carnobacterium, Enteroccus, Erysipelothrix, Gemella, Globicatella, Lactobacillus, Lactococcus, Bidobacteria, Leuconostoccocus, Pediococcus, Streptococcus, Tetragenococcus, or Bagococcus bacteria. In particular, the lactic acid bacterium can be a Lactococcus lactis. The DNA sequence encoding the xcex2-galactosidase can be selected from the group Streptococcus thermophilus, Lactobacillus bulgaricus, Bifobacterium species, Aspergillus niger, Aspergillus oryzae, Kluyveromyces fragilis, Kluyveromyces lactis, Bacillus subtillus or Arthrobacter species. The promoter can be a promoter from a gene that encodes an antimicrobial peptide, e.g., a lantibiotic, e.g., a nisin gene promoter, such as a nisA promoter. The bacterium can be permeabilized by an agent such as a chemical, a solvent (e.g., ethanol or isopropanol), or a detergent, (e.g., deoxycholate, sodium dodecyl sulfate, rhamnolipid, or chenodeoxycholate). In one embodiment of the method, hydrolysis of lactose is performed at 4xc2x0 C. In this embodiment, at least 90% of the lactose is hydrolyzed within 6 hours by a concentration of enzyme which is equivalent to 5000 o-nitrophenyl-xcex2-galactosidase (ONPG) units/liter. In another embodiment of the method, hydrolysis of lactose is performed at 55xc2x0 C. (or up to 63xc2x0 C). In this embodiment, at least 90% of the lactose is hydrolyzed within 2 hours by a concentration of enzyme equivalent to 5000 ONPG units/liter.
In another aspect, the invention features a method for hydrolyzing lactose including providing a permeabilized lactic acid bacterium containing a xcex2-galactosidase, wherein the bacterium exhibits a xcex2-galactosidase activity of at least about 4000 Miller units, and contacting the permeabilized bacterium with a liquid containing lactose, for a time sufficient to hydrolyze the lactose. The lactic acid bacterium can be Streptococcus, Aerococcus, Carnobacterium, Enteroccus, Erysipelothrix, Gemella, Globicatella, Lactobacillus, Lactococcus, Bidobacteria, Leuconostoccocus, Pediococcus, Streptococcus, Tetragenococcus, or Bagococcus. In particular, the lactic acid bacterium is a Lactococcus lactis. The xcex2-galactosidase can be encoded by a heterologous gene, e.g., a Streptococcus thermophilusxcex2-galactosidase. In other embodiments, the bacterium exhibits a xcex2-galactosidase activity of at least about 10,000 Miller Units.
In addition, the invention also features a permeabilized lactic acid bacterium containing a heterologous xcex2-galactosidase, e.g., a Streptococcus thermophilus xcex2-galactosidase, wherein the xcex2-galactosidase exhibits an activity of at least about 4000 Miller Units. The permeabilized bacterium can be Streptococcus, Aerococcus, Carnobacterium, Enteroccus, Erysipelothrix, Gemella, Globicatella, Lactobacillus, Lactococcus, Bidobacteria, Leuconostoccocus, Pediococcus, Streptococcus, Tetragenococcus, or Bagococcus. In particular, the lactic acid bacterium can be a Lactococcus lactis. The permeabilized bacterium can be in a lyophilized form, in a concentrated cell suspension, or immobilized. In one embodiment, the invention includes a composition including the permeabilized bacterium containing a heterologous xcex2-galactosidase which exhibits at least about 4000 Miller Units. In another embodiment, the invention includes a food product for use with a dairy product, wherein the food product includes the permeabilized bacterium containing a heterologous xcex2-galactosidase which exhibits an activity of at least about 4000 Miller Units.
Also within the invention is a method of administering, e.g., orally, lactase to a mammal, the method including administering to the mammal the permeabilized bacterium containing a heterologous xcex2-galactosidase which exhibits at least 4000 Miller Units.
The invention further features a reduced lactose dairy product including a dairy product, e.g., milk, and a permeablized Lactococcus lactis. The Lactococcus lactis can contain a Streptococcus thermophilus xcex2-galactosidase.
xe2x80x9cFood-grade bacteriaxe2x80x9d are microorganisms that are routinely consumed either as ingredients in fermented foods (e.g., cheese, bread, beer, yogurt) or as food or dietary supplements which aid in digestive processes (e.g. Lactobacilli, Bifidobacteria) and which have a record of being safe and non-toxic to consumers.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid which has a non-naturally occurring sequence, or which has the sequence of part or all of a naturally occurring gene but is free of, or differentially positioned with respect to the genes that flank the naturally occurring gene of interest in the genome of the organism in which the gene of interest naturally occurs. The term therefore includes a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote. It also includes a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment. It also includes a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein.
An xe2x80x9co-nitrophenyl-xcex2-galactosidase unit (ONPG unit)xe2x80x9d or xe2x80x9clactose unit (LU)xe2x80x9d is a concentration of xcex2-galactosidase which can hydrolyze a micromole of ONPG per minute, per liter of lactose containing liquid, e.g., milk or whey.
A first nucleic acid sequence that is xe2x80x9coperably linkedxe2x80x9d to a second nucleic acid sequence is one that is incorporated into a genetic construct so that the first nucleic acid sequence, e.g., an expression control sequence, effectively controls expression of the second nucleic acid sequence, e.g., a gene encoding xcex2-galactosidase.
A xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cproteinxe2x80x9d is any peptide-linked chain of amino acids, regardless of length or post-translational modification.
A xe2x80x9cheterologousxe2x80x9d nucleic acid sequence or protein is one that is not normally present in a given cell. For example, a gene such as LacZ from Streptococcus thermophilus (St. thermophilus), which is introduced into Lactoccocus lactis (L. lactis) is a heterologous LacZ in L. lactis. 
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which the amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine; arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,tyrosine, phenylalanine, tryptophan, histidine).
xe2x80x9cPercent sequence identityxe2x80x9d of two amino acid sequences or of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to an aggregation-disposed polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.